WO2003040695A2 - Protein knobs - Google Patents
Protein knobs Download PDFInfo
- Publication number
- WO2003040695A2 WO2003040695A2 PCT/US2002/035914 US0235914W WO03040695A2 WO 2003040695 A2 WO2003040695 A2 WO 2003040695A2 US 0235914 W US0235914 W US 0235914W WO 03040695 A2 WO03040695 A2 WO 03040695A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- protein
- knob
- subunit
- cysteine
- hcg
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the present invention relates to the field of protein tagging.
- purification currently involve fusing a tag to the carboxy- or amino- terminal ends of the protein or inserting
- the size of the tag usually must be relatively small (a few residues), unless the tag is inserted between protein
- cysteine residues which will require protection or the cysteine may become blocked and protein
- LHR lutropin receptor
- hCG appears to be secured largely
- hormone-receptor complex that differ radically, ⁇ Moyle, W. R., Campbell, R. , Rao, S. N. V., Ayad, N. G.,
- hormone contacts the concave surface of the receptor extracellular domain, ⁇ Jiang, X., Dreano, M,, Buckler, D.
- Protein-receptor interactions can be modeled on a computer, but
- modeling is a complex task, especially because molecules are flexible and adopt a number of conformations that are of a similar energy.
- the modeling of the protein-receptor binding process is also difficult because the
- mutations that change hCG activity may do so by altering key LHR contacts, shifting the positions of its
- proteins may be a required intermediate product of a scientific experiment or may be an end product.
- proteins must retain their biological activity.
- an invention typically requires that some of the initial contaminants have already been filtered. Accordingly, an invention
- knobs can be used to 'cover' specific surfaces on proteins. This type
- knobs could be used to block the activities of agents such as PTEN that promote apoptosis.
- the present invention relates to compositions comprising proteins tagged at a specific site
- a knob refers a tag for the protein which
- proteins tagged with a knob at a specific site in the protein are
- the site specific tagged protein contains a knob, a tail portion, and a protein portion.
- the tail portion is located
- the protein portion has a cysteine residue substituted for
- the tail portion may contain a protease or other cleavage
- knobs are disclosed. These methods involve selecting a desired protein for tagging, locating a specific site on
- the desired protein further comprises a tail portion
- knob contains a cysteine
- cysteine residue in the knob forms a disulfide bond with the cysteine residue in the
- tagged proteins of this invention are disclosed. These methods involve inserting a construct encoding a
- the encoded protein comprises a cysteine residue substituted at a desired site for
- tagging a tail portion, that has a cysteine residue and a cleavage site, at one end of the protein, and a knob
- the cell and purifying the protein based on the characteristics of the knob on the protein.
- the protein knobs are disclosed.
- the protein knobs may be used, for example, to map distances between proteins, probe
- Figure 1 Three-dimensional view of hCG. Illustration of the hCG ⁇ subunit residues that can
- the ⁇ tail is shown as a black ribbon.
- cysteine substitutions that enabled efficient crosslink between the ⁇ subunit residue and the probe cysteine
- the lighter gray spheres refer to residues that gave less amounts of crosslink.
- residues 90, 91 , and 92 appear to be too mobile to be seen in the crystal structure of hCG and the arbitrary
- Figure 3A Influence of cysteine substitutions in alpha subunit carboxyterminus. Binding of
- FIG. 4A Binding of hCG and hCG analogs with ⁇ tail attached to the ⁇ subunit residues in
- FIG. 4B cAMP accumulation of hCG or hCG analogs in which the ⁇ tail was attached to
- FIG. 5A Binding of hCG and hCG analogs with ⁇ tail attached to the ⁇ subunit residues at
- FIG. 5B cAMP accumulation of hCG or hCG analogs in which the ⁇ tail was attached to
- Figure 7 Amino acid sequences of the ⁇ -subunit and mutants having a substituted cysteine.
- Figure 8 Amino acid sequences of the ⁇ -subunit analogs. (Note, the cysteine substituted is in
- Figure 9A Protein Knob with the knob attached at the carboxyterminal end of the protein.
- Figure 9B Protein Knob with the knob attached at the aminoterminal end of the protein.
- Figure 10A Protein Knob where the knob is comprised of a cysteine residue.
- FIG. 10B Protein Knob where the knob is comprised of an amino acid sequence including
- cysteine residue fused to a protein.
- FIG. 10C Protein Knob where the cysteine residue of the knob is located on the surface of
- Figure 12 Summary of Activity of Crosslinked Chimera Analogs in LHR and FSHR Assays
- Figure 14A Effect of Knob on LH Receptor Signaling.
- Figure 14B Effect of Knob on LH Receptor Signaling.
- Figure 15 Amino acid sequences of other analogues (SEQ ID NO: 43 - SEQ ID NO: 53).
- Figure 16 Signal transduction and binding activities of LONG and SHORT heterodimers.
- Figure 17 Lutropin activity of hCG Analogs having ⁇ -lactamase knobs.
- Figure 18 cAMP accumulation in hCG or Analogs lacking ⁇ Asn52 oiigosaccharide.
- Figure 19 Binding of ⁇ K44A+hCG ⁇ to LHR.
- Figure 20A Binding of hCG and hCG analog ⁇ K44E, K45Q+hCG ⁇ to LHR.
- Figure 20B LHR cAMP response to hCG and ⁇ K44E, K45Q+hCG ⁇ .
- Figure 21 A Binding of hCG and hCG analog ⁇ K91 E+hCG ⁇ to LHR.
- Figure 21 B Relative activities of hCG and ⁇ K91 E+hCG ⁇ in LHR cAMP accumulation assays.
- Figure 22A LHR cAMP response to hCG and ⁇ K91 M+hCG ⁇ .
- Figure 22B LHR binding of hCG and ⁇ K91M+hCG ⁇ .
- Figure 23 LHR binding of hCG and analogs containing shortened linkers.
- Figure 24 LHR binding of hCG and an analog containing a shortened linker.
- Figure 25 Stimulation of LHR cAMP by hCG and ⁇ N52C+hCG ⁇ , S138C.
- Figure 26 LHR binding of analogs in which a truncated hCG ⁇ -subunit carboxyterminal tail
- Figure 27 LHR signaling of analogs in which a truncated hCG ⁇ -subunit carboxyterminal tail
- Figure 28 LHR binding of analogs in which a truncated hCG ⁇ -subunit carboxyterminal tail
- Figure 29 LHR signaling of analogs in which a truncated hCG ⁇ -subunit carboxyterminal tail
- Figure 30 LHR signaling of analogs in which a GGC tail was used to add a knob to ⁇ -subunit
- Figure 31 LHR signaling of analogs in which a truncated hCG ⁇ -subunit carboxyterminal tail
- Figure 31 LHR signaling of analogs showing the influence of the tail on adding a knob to hCG
- the terminal tags can be of any size, the tags inserted into a protein loop are usually limited to relatively few
- Proteins can also be tagged at
- the present invention provides for improved methods for labeling or tagging proteins.
- cysteine as described above, and allow various sized probes or knobs to be attached onto the surface of a cysteine, as described above, and allow various sized probes or knobs to be attached onto the surface of a cysteine, as described above, and allow various sized probes or knobs to be attached onto the surface of a cysteine, as described above, and allow various sized probes or knobs to be attached onto the surface of a cysteine, as described above, and allow various sized probes or knobs to be attached onto the surface of a
- the present invention provides for methods to add "knobs" of varying
- the knobs can be as small as a single cysteine residue.
- knobs can be a short peptide, such as residues surrounding hCG ⁇ -subunit residue 138.
- the knobs can also be a short peptide, such as residues surrounding hCG ⁇ -subunit residue 138.
- the knobs can also be a short peptide, such as residues surrounding hCG ⁇ -subunit residue 138.
- the knobs can also be a short peptide, such as residues surrounding hCG ⁇ -subunit residue 138.
- the knobs can also be a short peptide, such as residues surrounding hCG ⁇ -subunit residue 138.
- ⁇ -lactamase can be used as a knob.
- the knobs are added
- the present invention employs a new strategy for attaching a wide range of probes or tags to
- Knobs can be a
- fluorescent protein such as green fluorescent protein or related molecule. They can have the ability to bind
- antibody fragments sequences such as those found in the TAT protein of the human immunodeficiency virus
- composition comprising a protein portion, wherein the protein portion
- the protein portion and a knob, wherein the knob is located at the free terminal end of the tail portion and
- cysteine residue of the knob contains a cysteine residue, and wherein the cysteine residue of the knob forms a disulfide with the substituted
- protein portion refers to any protein or polypeptide.
- tail refers to any protein or polypeptide.
- portion refers to a stretch of amino acids of a sufficient length to permit the cysteine in the knob to form a
- the tail portion may comprise a native polypeptide
- the tail portion should comprise a non-native polypeptide added to a terminal end of the protein portion.
- knob also lack residues that will prevent the knob from attaching to the substituted cysteine, such as transmembrane domains or residues that will create a site for binding by other proteins.
- residues that will prevent the knob from attaching to the substituted cysteine such as transmembrane domains or residues that will create a site for binding by other proteins.
- the knob may include a single cysteine residue, a linear stretch of amino acids that
- cysteine contains a cysteine, a linear stretch of amino acids fused to a protein, where the cysteine is located in the
- a knob can be engineered
- the knob may be an
- epitope tag a signal sequence, a sequence with high specificity for a bead on a purification column, an
- the method involves selecting a desired protein, locating a site on the desired protein to be tagged,
- the method further involves
- the construct includes a cysteine residue substituted at the site to be tagged.
- construct refers to a nucleic acid vector comprising a promoter linked to an
- the construct further includes all
- sequences may include, but are not limited to, a promoter or
- RNA processing signals and/or a
- cassette refers to sequences required to ensure the RNA transcription and subsequent translation of the
- promoter refers to a DNA sequence that is
- RNA polymerase binds to RNA polymerase and is required to initiate RNA transcription of a gene.
- constructs of this invention may be modified by PCR and cassette mutagenesis to
- Knobs at specific sites on a protein can be used to probe the distance of the tested residues
- knobs can be utilized to determine the proximity
- knobs at specific sites Another advantage of using knobs at specific sites is that larger probes can be used to identify the
- the protein knobs can also be any desired site on the protein surface. Additionally, the protein knobs can also be any desired site on the protein surface. Additionally, the protein knobs can also be any desired site on the protein surface. Additionally, the protein knobs can also be any desired site on the protein surface. Additionally, the protein knobs can also be any desired site on the protein surface. Additionally, the protein knobs can also be any desired site on the protein surface. Additionally, the protein knobs can also be any desired site on the protein surface.
- protease recognition site is engineered within
- the tail can then be cleaved after the crosslink is accomplished, leaving the probe tethered by a
- proteases could be attached to either the aminoterminus or the carboxyterminus of a protein simply by fusing
- the protease may be held in a position that makes it incapable of
- the orientation of the protease may enable it to
- a protease may be engineered onto hCG
- This protease protein knob may be used in a therapeutically
- compositions and methods of this invention may be used in another embodiment of the invention.
- compositions and methods may be used to prepare and prepare a drink.
- DNA and is stabilized to the polymerase by a disulfide bond would be expected to stabilize the polymerase to
- compositions and methods of the invention are provided.
- glycoprotein hormones may be used to produce protein heterodimers of the glycoprotein hormones that lack one or more of their
- cysteine on the knob may be moved to a site on the surface of the knob
- knob portion This would permit the direct attachment of the knob to the protein in a desired orientation and
- the methods of this invention may be employed to tag proteins with
- Epitope tags are often attached to proteins to facilitate detection of protein-protein or protein-
- epitope tags were attached to the aminoterminal or carboxyterminal
- epitope tags added to the ends of proteins are decreased considerably when the ends of a protein are involved
- the methods of this invention permit the localization of epitope tags at sites other than
- aldehyde residues may be introduced at specific sites
- Aldehydes are very desirable reactive groups that are not normally found in proteins and can be
- residue can be introduced immediately after an enzyme cleavage site on a protein,
- a site recognized by enterokinase can be introduced immediately aminoterminal to a serine, which would be
- cysteine aminoterminal of the cysteine to be used to crosslink the knob to the target cysteine at the site to be tagged.
- tail portion amino acids refer to any tail portion amino acids and I, m, and n refer to the lengths of the tail portion amino acids.
- Cancer therapy may be an enzyme or a toxin that has the potential to kill cells until this activity is desired. Cancer therapy may be an enzyme or a toxin that has the potential to kill cells until this activity is desired. Cancer therapy may be an enzyme or a toxin that has the potential to kill cells until this activity is desired. Cancer therapy may be an enzyme or a toxin that has the potential to kill cells until this activity is desired. Cancer therapy may be an enzyme or a toxin that has the potential to kill cells until this activity is desired. Cancer therapy may be an enzyme or a toxin that has the potential to kill cells until this activity is desired. Cancer therapy may be an enzyme or a toxin that has the potential to kill cells until this activity is desired. Cancer therapy may be an enzyme or a toxin that has the potential to kill cells until this activity is desired. Cancer therapy may be an enzyme or a toxin that has the potential to kill cells until this activity is desired. Cancer therapy may be an enzyme or a toxin that has the potential to kill cells until this activity is desired. Cancer therapy may be
- cysteine to the aminoterminal or carboxyterminal end of the protein that has the ability to form a
- the knob may comprise a targeting protein fused to the end of the tail portion which would
- This strategy could be used to hide the activity of an enzyme or toxin until it reached a site
- disulfide would be designed to hold the knob in a position on the
- the present invention provides a method for precise separation of
- on the protein of interest may be at the carboxyterminal end, the aminoterminal end, or at any desired location
- the resulting protein-knob complex is run through a column.
- the protein-knob complex binds to the affinity resin and the unbound
- (Strategene's AffinityTM) pCAL vector may be used and the
- CBP calmodulin-bipding peptide
- the protein of interest may be any protein of interest.
- the protein knob has a short tail with a cleavage site.
- the knob tail must be short enough
- the knob is conducive for forming a disulfide bond with another protein.
- proteins with short tails would line up like a string of beads, connected by the disulfide bond between the
- cysteine on a first protein's knob and the cysteine on the protein portion of a second protein.
- the resulting chain of proteins may be run on a sucrose gradient and centrifuged, the chain would fall to
- the protein of interest may be constructed so that each
- protein knob interacts with other protein knobs to form a grid-like structure.
- the tails should be constructed so
- knob is unable to react with the cysteine on the protein portion, similar to the tails in the string of beads
- the proteins may also comprise tails and knobs at both terminal ends of the protein.
- portion should comprise multiple substitute cysteine residues so that knobs from more than one separate
- the proteins would form a grid-like structure as a single protein may form disulfide
- the present invention may be used to add cysteines to proteins. It is
- cysteine that is used to crosslink the knob to the cysteine that had been introduced at the site of the protein to
- the present invention may be used to create and obtain high
- heterodimer may comprise the protein portion. If the dimer of the protein portion does not have a naturally
- a tail portion may be fused to the dimer of the protein portion.
- the knob may comprise
- choriogonadotropin analogs implications for hormone folding and biological activity. Journal of Biological
- hFSH binds to the FSH receptor in a similar overall fashion but that different parts of hFSH make the key
- a 'tail portion' sequence requires both that the sequence be sufficiently
- cysteine in the tail portion can reach the cysteine on the surface of the protein to which it is
- lutropins such as hCG with the LH receptor
- follitropins such as hFSH with the FSH receptor
- hCG ⁇ -subunit residues 101-114 were replaced with their hFSH ⁇ -subunit counterparts, namely hFSH ⁇ -subunit residues 95-108
- heterodimers that were produced in COS-7 cells were quantified using a sandwich immunoassay
- COS-7 cells that were co-transfected with vectors encoding most of the ⁇ -subunit analogs
- glycosylation signal normally found at this position of hCG that is required for efficient secretion of the
- cysteines in the ⁇ -subunit analogs of analogs that formed little or no crosslinked heterodimers are at the
- cysteine in place of Tyr89. While this tyrosine is not essential for folding of the ⁇ -subunit because it can be
- hCG- ⁇ M47A an analog in which ⁇ Met47 had been replaced by alanine was nearly as active as hCG in
- replacing the ⁇ Lys51 sidechain may alter the conformation of the heterodimer.
- the ⁇ -subunit core can become crosslinked to the cysteine that was introduced into many of these ⁇ -subunit
- choriogonadotropin analogs implications for hormone folding and biological activity. Journal of Biological
- B111 ( ⁇ Y37C, ⁇ P40C, ⁇ L41C, ⁇ R42C, and ⁇ T86C) is nearest the B111 binding site
- the ⁇ -subunit to these sites may disrupt interactions between these portions of ⁇ -subunit loop 2 with the receptor or alter the conformation of ⁇ -subunit loop 2 in a way that reduces the ability of the hormone to
- portion of the ⁇ -subunit might not occupy the space in the groove between ⁇ -subunit loop 2 and ⁇ -subunit
- hCG- ⁇ S138C was expressed with several ⁇ -subunit analogs containing a cysteine in place of a residue
- ⁇ -subunit to this region alters the structure of the heterodimer or causes the end of the ⁇ -subunit to pass near
- ⁇ -lactamase a globular protein similar in size to hCG
- subunit loop 2 and ⁇ -subunit loops 1 and 3 does not participate in essential LH receptor contacts, a key requirement of an earlier model (Moyle, W, , Campbell, R. K., Rao, S. N, V., Ayad, N. G., Bernard, M. P.,
- Residues that have the greatest influence on hCG activity are located in the seatbelt, but even most
- a cDNA coding sequence for the hFSH ⁇ -subunit was obtained from Christie Kelton, Ares
- FC1-108 ⁇ Figure 8, SEQ ID NO: 41
- FC1-108 ⁇ lacking the leader peptide found in hFSH ⁇ - subunit is also illustrated in Figure 8.
- FC1-108 ⁇ to create FC1-108,S132C ⁇ ( Figure 8, SEQ ID NO: 42), FC1-108,S132C ⁇ was expressed with
- the heterodimers secreted by the cells into the culture media were measured using antibodies A113 and 125 l-
- the former is an antibody to the ⁇ -subunit and the latter is a monoclonal antibody that binds to hFSH
- the material was re-assayed with A113and 125 I-B603 as above to determine the amount of
- CFC101-114 ⁇ acid sequence of this chimera termed CFC101-114 ⁇ is shown in Figure 8 (SEQ ID NO: 38) minus the leader residues, An analog of CFC101-114 ⁇ in which residue Ser138 was converted to Cys was prepared by
- heterodimers secreted by the cells into the culture media were measured using antibodies A113 and 125 I-B110.
- the former is an antibody to the ⁇ -subunit and the latter is a monoclonal antibody that binds to hCG ⁇ -subunit.
- the material was re-assayed with A113and 125 I-B110 as above to determine the concentration of
- knob at ⁇ -subunit residue 37 was much more inhibitory on FSH receptor elicited cyclic AMP accumulation than
- Example 4 Knobs having ⁇ -lactamase.
- ⁇ -lactamase was chosen for use as a probe because its crystal
- lactamase is that it is an enzyme with a high turnover number that cleaves fluorescent substrates (Zlokarnik,
- ⁇ -lactamase has been used as an efficient reporter (Moore, J. T, Davis, S. T. & Dev, I. K. (1997) Anal. Biochem. 247, 203-209), albeit in a very different context than is envisioned here, ⁇ -lactamase is also inhibited
- the ⁇ -lactamase knob further simply by adding the inhibitor.
- the SHORT protein has only one amino acid between the coupling cysteine and the start of the ⁇ -
- cysteine could serve as the coupling cysteine, a
- ⁇ -lactamase knob to this site can offset the loss in efficacy caused by removal of the oligosaccharide from loop 2.
- the relatively high activity of both these LONG and SHORT analogs show that, ⁇ -subunit residue is not
- Example 5 Knobs having a coupling cysteine at the aminoterminal end of the ⁇ -subunit.
- a construct capable of expressing the protein may be prepared such that a cysteine residue is substituted for the native residue at the
- the construct is then inserted into a cell for expression resulting in the
- Addition of a knob to a protein involves introducing a cysteine onto the surface where the knob
- cysteine in the end of the protein can form a disulfide with that that has been added to the
- cysteine to be stabilized at the surface creating a knob at this site (see Figure 9).
- composition of the tail portion can be varied extensively
- knobs larger the knob.
- the smallest knobs would consist only of a cysteine. (See Figure 10A). These would be
- the knob can be increased in at least three fashions.
- a protein can be fused to the end of the protein used to construct the knob. (See Figure 10B). As described above, ⁇ -lactamase was fused to the end of the knob.
- ⁇ -lactamase is not the only protein that is suitable for this purpose.
- the choice of ⁇ -lactamase is not the only protein that is suitable for this purpose. The choice of ⁇ -lactamase
- lactamase as a probe was facilitated by its crystal structure, which showed that its aminoterminal end was
- distance between the coupling cysteine and the fusion protein probe can be varied by increasing or reducing
- Example 7 Use of shortened tail portion as the probe.
- the tail portion itself can also be used to probe the surface of proteins and there
- these include those in which it would be desirable to cover a portion of an active site of a protein,
- this groove formed a key receptor contact.
- the use of the tail portion described earlier would permit it to pass
- hCG ⁇ , ⁇ 116-135,S138C or hCG ⁇ , ⁇ 121-135,S138C had substantial receptor binding activity.
- toxin site would permit preparation and use of reagents capable of entering cells where endogenous or other proteases would cleave the tail portion to activate the toxin. These agents would be useful as
- Example 8 Use of probes attached to the ⁇ -subunit.
- the alpha subunit were produced that contained the carboxyterminal residues in the hCG ⁇ -subunit. While it
- LHR LH receptors
- the knob reduced receptor interaction more when it
- carboxyterminal end of the ⁇ -subunit is near the small seatbelt loop after the hormone interacts with the LHR.
- ⁇ -subunit can be attached to several sites in the ⁇ -subunit, indicating that its location is not constrained
- compositions along with the methods and procedures described herein are presently representative of
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Abstract
Description
Claims
Priority Applications (11)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP02789525A EP1497634A4 (en) | 2001-11-08 | 2002-11-08 | Protein knobs |
KR10-2004-7006994A KR20040063928A (en) | 2001-11-08 | 2002-11-08 | Protein knobs |
EA200400647A EA200400647A1 (en) | 2001-11-08 | 2002-11-08 | MARK OF PROTEINS |
MXPA04004289A MXPA04004289A (en) | 2001-11-08 | 2002-11-08 | Protein knobs. |
CA002466595A CA2466595A1 (en) | 2001-11-08 | 2002-11-08 | Protein knobs |
IL16180402A IL161804A0 (en) | 2001-11-08 | 2002-11-08 | Protein knobs |
JP2003542901A JP2005509652A (en) | 2001-11-08 | 2002-11-08 | Protein knob |
US10/797,553 US20060177894A1 (en) | 2002-11-08 | 2004-03-10 | Protein knobs |
HR20040242A HRP20040242A2 (en) | 2001-11-08 | 2004-03-12 | Protein knobs |
ZA2004/04373A ZA200404373B (en) | 2001-11-08 | 2004-06-03 | Protein knobs |
NO20042358A NO20042358L (en) | 2001-11-08 | 2004-06-07 | Protein knobs |
Applications Claiming Priority (2)
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---|---|---|---|
US34528301P | 2001-11-08 | 2001-11-08 | |
US60/345,283 | 2001-11-08 |
Publications (2)
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WO2003040695A2 true WO2003040695A2 (en) | 2003-05-15 |
WO2003040695A3 WO2003040695A3 (en) | 2004-11-11 |
Family
ID=23354368
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2002/035914 WO2003040695A2 (en) | 2001-11-08 | 2002-11-08 | Protein knobs |
Country Status (13)
Country | Link |
---|---|
EP (1) | EP1497634A4 (en) |
JP (2) | JP2005509652A (en) |
KR (1) | KR20040063928A (en) |
CN (1) | CN1596304A (en) |
CA (1) | CA2466595A1 (en) |
EA (1) | EA200400647A1 (en) |
HR (1) | HRP20040242A2 (en) |
IL (1) | IL161804A0 (en) |
MX (1) | MXPA04004289A (en) |
NO (1) | NO20042358L (en) |
PL (1) | PL373566A1 (en) |
WO (1) | WO2003040695A2 (en) |
ZA (1) | ZA200404373B (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090053223A1 (en) * | 2004-07-16 | 2009-02-26 | . | Expression-enhanced polypeptides |
CN112986571A (en) * | 2019-12-12 | 2021-06-18 | 中国科学院大连化学物理研究所 | Method for identifying Midkine spatiotemporal network interaction protein |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
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GB201403815D0 (en) * | 2014-03-04 | 2014-04-16 | Mologic Ltd | Assay |
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DK1775346T3 (en) * | 1997-06-25 | 2009-09-28 | Merck Serono Sa | Disulfide crosslinked glycoprotein hormone analogues, their preparation and use |
-
2002
- 2002-11-08 CN CNA02822101XA patent/CN1596304A/en active Pending
- 2002-11-08 EA EA200400647A patent/EA200400647A1/en unknown
- 2002-11-08 PL PL02373566A patent/PL373566A1/en not_active Application Discontinuation
- 2002-11-08 JP JP2003542901A patent/JP2005509652A/en active Pending
- 2002-11-08 CA CA002466595A patent/CA2466595A1/en not_active Abandoned
- 2002-11-08 MX MXPA04004289A patent/MXPA04004289A/en not_active Application Discontinuation
- 2002-11-08 EP EP02789525A patent/EP1497634A4/en not_active Withdrawn
- 2002-11-08 KR KR10-2004-7006994A patent/KR20040063928A/en not_active Application Discontinuation
- 2002-11-08 IL IL16180402A patent/IL161804A0/en unknown
- 2002-11-08 WO PCT/US2002/035914 patent/WO2003040695A2/en active Application Filing
-
2004
- 2004-03-12 HR HR20040242A patent/HRP20040242A2/en not_active Application Discontinuation
- 2004-06-03 ZA ZA2004/04373A patent/ZA200404373B/en unknown
- 2004-06-07 NO NO20042358A patent/NO20042358L/en not_active Application Discontinuation
-
2007
- 2007-10-11 JP JP2007265961A patent/JP2008110973A/en active Pending
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090053223A1 (en) * | 2004-07-16 | 2009-02-26 | . | Expression-enhanced polypeptides |
US8518403B2 (en) * | 2004-07-16 | 2013-08-27 | Amgen Research (Munich) Gmbh | Expression-enhanced polypeptides |
CN112986571A (en) * | 2019-12-12 | 2021-06-18 | 中国科学院大连化学物理研究所 | Method for identifying Midkine spatiotemporal network interaction protein |
CN112986571B (en) * | 2019-12-12 | 2023-04-11 | 中国科学院大连化学物理研究所 | Method for identifying Midkine spatiotemporal network interaction protein |
Also Published As
Publication number | Publication date |
---|---|
HRP20040242A2 (en) | 2005-02-28 |
JP2005509652A (en) | 2005-04-14 |
ZA200404373B (en) | 2005-11-30 |
JP2008110973A (en) | 2008-05-15 |
WO2003040695A3 (en) | 2004-11-11 |
CA2466595A1 (en) | 2003-05-15 |
NO20042358L (en) | 2004-08-09 |
CN1596304A (en) | 2005-03-16 |
MXPA04004289A (en) | 2005-03-31 |
KR20040063928A (en) | 2004-07-14 |
PL373566A1 (en) | 2005-09-05 |
EA200400647A1 (en) | 2004-10-28 |
EP1497634A4 (en) | 2007-02-07 |
EP1497634A2 (en) | 2005-01-19 |
IL161804A0 (en) | 2005-11-20 |
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